Fusion protein comprising Anti-c-met antibody and vegf-binding fragment

a technology of vegf and anticmet, which is applied in the field offusion protein comprising anticmet antibody and vegfbinding fragment, can solve the problems of poor prognosis of most patients with over-expressed c-met, and achieve the effect of improving anticancer and anti-angiogenesis effects

Active Publication Date: 2014-10-02
SAMSUNG ELECTRONICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0014]Still another embodiment provides a method of preparing a bispecific antibody targeting c-Met and VEGF and having i

Problems solved by technology

In addition, c-Met is over-expressed in cancer cells, and thereby widely implicated in cancer incidence, cancer metastasis, cancer cell migration, cancer cell invasio

Method used

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  • Fusion protein comprising Anti-c-met antibody and vegf-binding fragment
  • Fusion protein comprising Anti-c-met antibody and vegf-binding fragment
  • Fusion protein comprising Anti-c-met antibody and vegf-binding fragment

Examples

Experimental program
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Effect test

reference example 1

Construction of Anti-c-Met Antibody

[0128]1.1. Production of “AbF46”, a Mouse Antibody to c-Met

[0129]1.1.1 Immunization of Mouse

[0130]To obtain immunized mice necessary for the development of a hybridoma cell line, each of five BALB / c mice (Japan SLC, Inc.), 4 to 6 weeks old, was intraperitoneally injected with a mixture of 100 μg of human c-Met / Fc fusion protein (R&D Systems) and one volume of complete Freund's adjuvant. Two weeks after the first injection, a second intraperitoneal injection was conducted on the same mice with a mixture of 50 μg of human c-Met / Fc protein and one volume of incomplete Freund's adjuvant. One week after the second immunization, the immune response was finally boosted. Three days later, blood was taken from the tail and the sera were 1 / 1000 diluted in PBS and used to examine a titer of antibody to c-Met by ELISA. Mice found to have a sufficient antibody titer were selected for use in the cell fusion process.

[0131]1.1.2. Cell Fusion and Production of Hybr...

example 1

Preparation of Fusion Proteins

[0181]The anti-c-Met antibodies manufactured in Reference Example 1 were fused to a linker by coupling the C-terminal of their heavy chain therewith. Thereafter, an Ig2 domain, that is, amino acids from 129th to 229th among the amino acids constituting VEGF receptor 1 was sequentially fused at the terminal of the linker to manufacture antibodies capable of binding to c-Met and VEGF at the same time (see FIG. 1).

[0182]Among 1338 amino acids constituting VEGF receptor 1 (P17948.2; SEQ ID NO: 109), a gene sequence encoding 101 amino acids from 129th to 229th constituting the Ig2 domain which has been shown to be most important for VEGF-binding was secured from NCBI database.

Ig2 domain (VIG2) amino acid sequence (SEQ ID NO: 110):SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIg2 domain (VIG2) nucleotide sequence (SEQ ID NO: 111):AGTGATACAGGTAGACCTTTCGTAGAGATGTACAGTGAAATCCCCGAAATTATACACATGACTGAAGGAAGGGAGC...

example 2

c-Met Target and Degradation Activity of Fusion Proteins

[0187]2.1. Affinity Test

[0188]The binding kinetics of the double-targeted fusion proteins manufactured in the above were analyzed using Biacore T100 instrument (GE Healthcare). A human Fab binding agent (GE Healthcare) was immobilized on the surface of a CM5 chip (GE Healthcare, Cat. No. BR-1005-30) according to the manufacturer's manual. About 90120 RU of L3-1Y or MV10AY was captured and various concentrations of c-Met (R&D Systems, Cat. No. 358-MT / CF) were introduced to the captured antibodies by injection. Then, 10 mM Glycine-HCl (pH 1.5) solution was added thereto, whereby the surface was regenerated. In order to measure the binding kinetics, the data obtained from the above experiment was fitted using BIAevaluation software (GE Healthcare, Biacore T100 evaluation software).

[0189]The results obtained by using the BIAevaluation software are shown in Table 4 below.

TABLE 4SampleRmax (RU)KD (nM)ka (1 / Ms)kd (1 / s)L3-1Y89.637.7 × ...

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Abstract

There are provided a fusion protein formed by coupling of anti-c-Met antibody and VEGF-binding fragment, a bispecific antibody comprising the fusion protein, a polynucleotide encoding the fusion protein, a transformant comprising the polynucleotide, a pharmaceutical composition comprising the bispecific antibody as an active ingredient, and a method for preparing the bispecific antibody which is targeted at c-Met and VEGF at the same time, with improved anticancer and anti-angiogenesis effects, comprising coupling an anti-c-Met antibody with a VEGF-binding fragment.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims the benefits of Korean Patent Application No. 10-2013-0033872, filed on Mar. 28, 2013, in the Korean Intellectual Property Office, the entire disclosure of which is hereby incorporated by reference.INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY[0002]Incorporated by reference in its entirety herein is a computer-readable nucleotide / amino acid sequence listing submitted concurrently herewith and identified as follows: One 160,391 Byte ASCII (Text) file named “715587_ST25.TXT,” created on Mar. 27, 2014.BACKGROUND OF THE INVENTION[0003]1. Field[0004]Provided are a fusion protein comprising an anti-c-Met antibody and a VEGF-binding fragment which are coupled to each other, a bispecific antibody comprising the fusion protein, a polynucleotide encoding the fusion protein, a transformant comprising the polynucleotide, a pharmaceutical composition comprising the bispecific antibody as an active ingredient, ...

Claims

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Application Information

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IPC IPC(8): C07K16/32C07K16/22
CPCC07K16/22C07K16/32A61K2039/505A61P3/10A61P9/10A61P27/02A61P35/00A61P43/00C07K14/71C07K16/2863C07K2317/10C07K2317/24C07K2317/515C07K2317/52C07K2317/53C07K2317/56C07K2317/565C07K2317/622C07K2317/75C07K2317/76C07K2317/92C07K2319/00A61K39/395C07K16/46
Inventor SONG, YUN JEONGSONG, HO YEONGLEE, SEUNG HYUNKIM, KYUNG AHJEONG, YUN JU
Owner SAMSUNG ELECTRONICS CO LTD
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